This research focuses on studies of viral and cellular pathogenesis of human hepatocellular carcinoma (HCC) one of the most common and devastating malignant tumors in the world. During the previous grant period, we have shown that generation of hepatitis B virus (HBV) variants occurs during active replication and these molecular events may lead to viral persistence in the liver. We now wish to define their role in the development of HCC and 1) assess the biological properties of HBV variants, determine if HBV variant forms integrate into tumor DNA and investigate the cellular proteins which interact with the viral HBx transactivator and may be important in the regulation of growth control genes in the liver. In addition, previous investigations by us have demonstrated that the insulin receptor substrate-1 protein (IRS-1), a substrate for tyrosine kinase containing growth factor receptors, is overexpressed in HBV related human HCC. Furthermore, preliminary studies presented herein suggest that simple three to four fold overexpression of IRS-1 leads to cellular transformation and activates the growth factor mediated signal transduction cascade. In this regard we will 2) determine if IRS-1 and another protein namely p36 protein kinase substrate are overexpressed in HCC tumors derived from man and various experimental animal models and represent general features of mammalian hepatocyte transformation. The concentration of these molecules in early preneoplastic lesions in the liver will also be assessed during the natural history of tumor development. At the molecular level, other experiments will evaluate functional domains of the IRS-1 molecule and determine if expression of IRS-1 and mutant constructs with Y to F changes in tile YVNI and YMXM motifs that alter the binding of GRB2 and PtdIns-3- kinase proteins respectively, leads to malignant transformation through activation of the various known cellular pathways associated with the growth factor mediated signal transduction cascade. In addition, the functional significance of the Syp binding site for a tyrosine phosphatase and the pleckstrin homology (PH) G-beta-gamma binding domain of IRS-1 protein will be explored since little is known regarding their role in cellular transformation. Finally, we have shown that the 5' upstream regulatory region of the IRS-1 gene contains AP-1 and AP-2 transcription factor binding sites. Growth control genes with such regulatory regions have been found to be upregulated by the HBx transactivator protein. Thus, we plan studies to possibly link viral and cellular factors and 3) determine if the HBx transactivator protein, along with its recently identified cellular partners, upregulate IRS-1 gene expression. These efforts will provide new information on the viral and cellular mechanisms of hepatocarcinogenesis and the role of overexpression of genes such as IRS-1 and p36 during this process.